Method of producing IgG

ABSTRACT

A method for producing IgG from plasma for medical applications, comprising at least: (i′) removal of albumin resulting in an IgG fraction, (ii′) purifying IgG from an IgG fraction, which is derived from the IgG fraction obtained in step (i′), by adsorbing IgG to a cation exchanger and collecting the adsorbed IgG fraction, and (iii′) virus inactivation in an IgG fraction derived from the IgG fraction collected in step (ii′). The method is characterized in; (I) concentrating the IgG fraction obtained in step (i′), (II) adjusting pH to 4±0.1 in the IgG fraction released from the cation exchanger in step (ii′), and preferably maintaining the pH below 6.0 during the remaining steps of the method; and (III) carrying out the virus inactivation (step iii′) by using chemicals at a temperature of 30° C.±2° C. for at least 4 hours. Anticomplementary activity is typically below 1 CH 50 /mg immunoglobulin.

FIELD OF THE INVENTION

The present invention relates to a method of producing IgG for medicalapplications. The method of the invention provides an IgG product havinglow anticomplementary activity (ACA).

BACKGROUND OF THE INVENTION

IgG prepared from human plasma is widely used in the treatment ofagammaglobulinaemia, idiopathic thrombocytopenic purpura and in theprophylaxis of certain diseases. IgG preparations are administeredintramuscularly as well as intravenously.

Within the field of the art it is well known that isolated IgGpreparations have marked anticomplementary activities (ACA). It has beenshown that the components responsible for these activities areaggregates of IgG that form either spontaneously or as a result of theisolation procedure. These anticomplementary aggregates have been shownto be harmful in several clinical applications of the IgG products. Forexample, intravenous administration of IgG preparations can give rise toadverse side reactions, including anaphylactic shock.

Several solutions have been proposed to overcome the problem with ACA inIgG preparations. For example, in U.S. Pat. No. 3,966,906 a process isdescribed for treating a crude gamma globulin fraction of serum withpepsin to disaggregate IgG and reduce anticomplement activity. However,the therapeutic effect provided by such a preparation is unacceptablyshort since it is rapidly excreted. Another drawback with the pepsintreated immunoglobulins is that their Fc binding capacity is lower thanfor native immunoglobulins.

Attempts have been made to stabilise pepsin treated IgG preparations,such as by polyethylene glycol (PEG) see for example WO 86/06993.

To solve the problem with high ACA activity it has been proposed tochemically modify the IgG preparations. For example, in U.S. Pat. No.3,902,262 a portion of the disulphide linkages of the IgG molecule isreduced to —SH groups and then the —SH groups are alkylated.

For obvious reasons, it would be desirable to have an IgG product whichis free from enzymatic and other chemical modification and to be asclose to native as possible. A method fulfilling these criteria has beendescribed in “An improved chromatographic method for production of IgGfrom human plasma” by I. Andersson, L-O Lindquist, J. Berglöf, presentedat the “XXIII Congress of the ISBT”, Amsterdam, The Netherlands, Jul.2-8, 1994). However, this procedure also shows unsatisfactory highACA-levels and does not fulfil the FDA and EU requirements forintravenous drugs.

Known methods for the manufacture of IgG compositions typically compriseseveral steps selected amongst:

a) buffering plasma, for instance either by subjecting the plasma to anappropriate gel filtration chromatography or by diafiltration;

b) removing euglobulins, such as by precipitation;

c) removing the albumin fraction (albumin), for instance by bindingalbumin and the like to an anion exchanger leaving the IgG in theunbound fraction (IgG fraction);

d) purifying, after removal of euglobulins and albumin, the IgG fractionobtained in step (c) on an anion exchanger and collecting the unboundfraction;

e) purifying the plasma fraction obtained in step (d) on a cationexchanger and collecting the bound fraction (adsorption and release ofIgG);

f) concentrating the IgG enriched plasma fraction obtained in step (e)(IgG released from the cation exchanger), preferably by ultrafiltration;

g) inactivating viruses by adding virus inactivation chemicals,preferably a solvent/detergent (S/D) solution, to an IgG enriched plasmafraction, for instance the fraction obtained in step (f);

h) removing the virus antiviral chemicals added in step (g), preferablyby adsorbing IgG to a cation exchanger and releasing and collecting thebound fraction;

i) concentrating the bound fraction collected in step (h), for instanceby ultrafiltration;

j) formulating of the fraction concentrated in step (i);

k) sterile filtration of the formulated IgG obtained in step (k).

SUMMARY OF THE INVENTION

The present invention provides a solution for producing IgG productshaving reduced ACA, by modifying earlier known methods.

Thus in a first aspect the invention relates to a method of producingIgG from plasma for medical applications, comprising at least: (i′)removal of albumin resulting in an IgG fraction, (ii′) purifying IgGfrom an IgG fraction, which is derived from the IgG fraction obtained instep (i′), by adsorbing IgG to a cation exchanger and collecting theadsorbed IgG fraction, and (iii′) virus inactivation in an IgG fractionderived from the IgG fraction collected in step (ii′). The method ischaracterized in

(I) concentrating the IgG fraction obtained in step (i′),

(II) adjusting pH to 4±0.1 of the IgG fraction released from the cationexchanger used in step (ii′), and preferably maintaining the pH below6.0 during the remaining steps of the method; and

(III) carrying out the virus inactivation (step iii′) by using virusinactivation chemicals, preferably a solvent/detergent (S/D) solution,at a temperature of 30° C.±2° C. for at least 4 hours.

Adjustment of pH to around 4 in (II) permits virus inactivation to becarried out at around 30° C. In the corresponding earlier process, inwhich pH was 5,5, the level of the proteolytic activity at 30° C. wasunacceptable.

A preferred variant of the process according to the invention comprisessteps (a)-(k) above in which the albumin removal step (c) corresponds to(i′), the cation exchange step (e) corresponds to (ii′), and the virusinactivation (g) corresponds to (iii′).

The purification and/or removal steps above are preferably run aschromatography. Appropriate separation media used in these steps arehydrophilic in the sense that they are able to expose surfaces carryinghydrophilic groups, such as hydroxy, amido etc., to the liquid samplecontaining IgG. Appropriate separation media may be found amongst thosethat are based on synthetic polymers and/or biopolymers (for instancepolysaccharides) carrying hydrophilic groups, as referred to above.Depending on where in the process the media is to be applied they may beuncharged, or may carry positively charged (e.g. ammonium groups) and/ornegatively charged groups (e.g. carboxy groups and sulphonic acidgroups). The following chromatographic media are preferred:

step a): Sephadex G25;

step c): DEAE Sepharose FF;

step d): Q Sepharose FF;

step e): CM Sepharose FF;

step h): CM Sepharose FF.

Sephadex and Sepharose (Amersham Pharmacia Biotech AB, Uppsala, Sweden)are based on cross-linked dextrand and agarose, respectively. DEAE meansthat the base matrix (cross-linked dextran is substituted withdiethylaminoethyl groups. Analogously Q stands for quaternary ammoniumgroups, and CM for carboxy methyl groups.

The concentrating according to (I) is preferably performed immediatelyafter albumin removal (for instance after step c as defined above) byultrafiltration, to less than or equal to the volume of the startingplasma.

To be able to use an acetate buffer in the step for removal of virusinactivation chemicals (step h), the ionic strength is adjusted to about1.40 mS before this step.

In a preferred embodiment, the method also comprises, in step (i)lowering of ionic strength to 0.5 mS±0.1, preferably by diafiltrationagainst distilled water.

DETAILED DESCRIPTION OF THE INVENTION

Below the invention will be described in more detail in association withthe accompanying drawing, FIG. 1, which schematically shows a preferredembodiment of the method according to the invention, wherein thecharacterising features of the method as defined in the claims are inbold letters.

Starting Material

Both Recovered Plasma, Fresh Frozen Plasma and Source plasma can be usedas starting material.

The following subclasses of plasma can be identified and used asstarting material.

1. Cryosupernatant plasma that has passed a chromatographic step foradsorption of vitamin K dependent factors (FIX, prothrombin, FVII, FX).

2. Cryosupernatant plasma from which the prothrombin complex has notbeen removed.

3. Plasma that has passed a gel filtration medium (preferably havingexclusion limits in the same range as Sepharose 4FF (Amersham PharmaciaBiotech AB, Uppsala, Sweden) for removal of FVIII and that has passed achromatographic step for adsorption of vitamin K dependent factors (FIX,prothrombin, FVII, FX).

4. Plasma that has passed a gel filtration medium for removal of FVIIIand from which the prothrombin complex has not been removed.

The gel filtration medium preferably exclusion limits in the same rangeas Sepharose FF (Amersham Pharmacia Biotech AB).

In addition the starting material could also be free from ATIII and/orFibrinogen.

Below two non-limiting Examples of methods according to the invention topurify IgG are described. The methods are performed at room temperature,if nothing else is stated.

Anticomplementary activity (ACA) refers to measurements in the finalproduct, is measured according to Eur. Pharmacopoeia Monograph (1997)page 963 (2.6.17) and should not be higher than 1 CH₅₀/mgimmunoglobulin.

EXAMPLE 1

625 L of thawed plasma is buffer exchanged into a 0.005 M NaAc (sodiumacetate) pH 7 on a column of diameter Di=800 mm and bed height of H=600mm, packed with Sephadex G-25 C. The flow is more than 100 cm/h,preferably 300 cm/h corresponding to a flow rate of 1500 L/h.

The eluted plasma is collected in a tank and 1 M acetic acid is addedduring stirring until the pH 5.2 is reached. The plasma is left standingwithout stirring for 4-12 hours at a temperature of 4-10° C. Afterstanding the formed euglobulin precipitation is removed bycentrifugation.

The plasma is adjusted with 1M NaAc, pH 5.2 to a final ionic strength ofI=1.4 mS (Range I=1.30-1.50). The pH shall be between 5.15-5.25.

The plasma is applied in 6 cycles, 25-30 g of albumin per liter gel, ona column of Di=1000 mm and H=150 mm, packed with DEAE Sepharose FF andequilibrated with 0.020 M NaAc, pH 5.2. The linear flow rate is morethan 60 cm/h, preferably 120 cm/h corresponding to a flow rate of 942L/h. In the equilibration buffer the IgG will pass the column whilst thealbumin is adsorbed. After 3 cycles the column is washed with 1.7 Vc(Vc=bed volume) of 0.15 M NaAc pH 4.0+0.5 M NaCl, 0.5 Vc of 0.5 M NaOHand 1.7 Vc of 0.15 M NaAc pH 4.0.

The IgG fraction of about 2350 L is concentrated by ultrafiltration to afinal volume less or equal to 625 L, preferably 400-500 L. The procedureshall be started at the latest, when the whole fraction is collectedfrom the DEAE Sepharose column.

The IgG solution is pH adjusted to pH 6.5 (6.45-6.55) with 1M NaOH andthe ionic strength is adjusted to 1.40 mS (1.30-1.50 mS) by adding ofWFI water (WFI=water for injection).

The IgG solution is applied in 6 cycles on a column of Di=1000 mm andH=150 mm, packed with Q Sepharose FF and equilibrated with 0.020 M NaAc,pH 6.5. Linear flow rate is more than 30 cm/h preferably 100 cm/hcorresponding to 785 L/h. After 3 cycles the column is washed with 0.5Vc of 0.5 M NaOH and 1.7 Vc of 0.15 M NaAc pH 4.0 The break throughfraction containing IgG is directly adsorbed on the next column ofDi=800 mm and H=80 mm and packed with CM Sepharose FF. When the IgGfraction from all 6 cycles has been pumped through, the column is washedwith 10 Vc of 0.01 M Glycine buffer, pH 7.0. The IgG is then eluted with7 Vc of 0.1 M Glycine+0.15 M NaCl pH 9.0.

The pH of the solution is adjusted to 4.0±0.1 with 1M HAc (Acetic acid)and concentrated by ultrafiltration to about 5% IgG.

Virus inactivation chemicals, Triton X-100 and TNBP, are added to theIgG solution during stirring. This mixture is transferred to theincubation tank for heat treatment at 30° C.±2° C. for 4-16 hours. Theionic strength of the solution is adjusted to 1.40 mS by dilution withWFI and applied in 1 cycle on another column of Di=800 mm and H=80 mm,packed with CM Sepharose FF and equilibrated with 0.020 M NaAc buffer pH4.0. The linear flow rate is more than 40 cm/h, preferably 80 cm/h,corresponding to 400 L/h. After application the column is washed with 10Vc of 0.01 M Glycine buffer pH 7.0 in order to remove the inactivationchemicals. The IgG is eluted with 7 Vc of 0.1 M Glycine+0.15 M NaCl pH9.0 at the same flow rate and adjusted to pH 4.0 with 1M HCl. Thesolution is then concentrated by ultrafiltration to 5% to 7% IgG and theionic strength is adjusted by diafiltration to 0.5 mS±0.2 mS. Finallythe solution is adjusted to 5.0%.

The solution is formulated to the following composition:

Sucrose 1 g/g IgG

IgG 5%

pH 4.0.

ionic strength 0.5 mS±0.2 mS

After sterile filtration, filling and capping the solution is ready fordelivery or storage.

ACA for different batches measured as defined above was found to be0.5-0.7 CH₅₀/mg immunoglobulin.

EXAMPLE 2

625 L of thawed plasma is buffer exchanged into a 0.005 M NaAc (sodiumacetate) pH 7 on a column of diameter Di=800 mm and bed height of H=600mm, packed with Sephadex G-25 C. The flow is more than 100 cm/h,preferably 300 cm/h corresponding to a flow rate of 1500 L/h.

The eluted plasma is collected in a tank and 1M acetic acid is addedduring stirring until the pH 5.2 is reached. The plasma is left standingwithout stirring for 4-12 hours at a temperature of 4-10° C. Afterstanding the formed euglobulin precipitation is removed bycentrifugation.

The plasma is adjusted with 1M NaAc, pH 5.2 to a final ionic strength ofI=1.4 mS (Range I=1.30-1.50). The pH shall be between 5.15-5.25.

The plasma is applied in 6 cycles, 25-30 g of albumin per liter gel, ona column of Di=1000 mm and H=150 mm, packed with DEAE Sepharose FF andequilibrated with 0.020 M NaAc, pH 5.2. The linear flow rate is morethan 60 cm/h, preferably 120 cm/h corresponding to a flow rate of 942L/h. In the equilibration buffer the IgG will pass the column whilst thealbumin is adsorbed. After 3 cycles the column is washed with 1.7 Vc(Vc=bed volume) of 0.15 M NaAc pH 4.0+0.5 M NaCl, 0.5 Vc of 0.5 M NaOHand 1.7 Vc of 0.15 M NaAc pH 4.0.

The IgG fraction of about 2350 L is concentrated by ultrafiltration to afinal volume less than or equal to 625 L, preferably 400ñ500 L. Theprocedure shall be started at the latest, when the whole fraction iscollected from the DEAE Sepharose column.

The IgG solution is pH adjusted to pH 6.5 (6.45-6.55) with 1M NaOH andthe ionic strength is adjusted to 1.40 mS (1.30-1.50 mS) by adding ofWFI water (WFI=water for injection).

The IgG solution is applied in 4 cycles on column of Di=1000 mm andH=150 mm, packed with a mixed bed of DEAE Sepharose FF and ArginineSepharose FF in a proportion 60%/40% and equilibrated with 0.020 M NaAc,pH 6.5. Linear flow rate is more than 30 cm/h, preferably 100 cm/hcorresponding to 785 L/h. After 2 cycles the column is washed with 0.5Vc of 0.5 M NaOH and 1.7 Vc of 0.15 M NaAc pH 4.0. The break throughfraction containing IgG is directly adsorbed on the next column ofDi=800 mm and H=80 mm, packed with CM Sepharose FF. When the IgGfraction from all 6 cycles has been pumped through, the column is washedwith 10 Vc of 0.01 M Glycine buffer, pH 7.0. The IgG is then eluted with7 Vc of 0.1 M Glycine+0.15 M NaCl pH 9.0.

The pH of the solution is adjusted to 4.0±0.1 with 1M HAc (Acetic acid)and concentrated by ultrafiltration to about 5% IgG.

Virus inactivation chemicals, Triton X-100 and TNBP, are added to theIgG solution during stirring. This mixture is transferred to theincubation tank for heat treatment at 30° C.±2° C. for 4-16 hours. Theionic strength of the solution is adjusted to 1.40 mS by dilution withWFI and applied in 1 cycle on another column of Di=800 mm and H=80 mm,packed with CM Sepharose FF and equilibrated with 0.020 M NaAc buffer pH4.0. The linear flow rate is more than 40 cm/h, preferably 80 cm/hcorresponding to 400 L/h. After application the column is washed with 10Vc of 0.01 M Glycine buffer pH 7.0 in order to remove the inactivationchemicals. The IgG is eluted with 7 Vc of 0.1 M Glycine+0.15 M NaCl pH9.0 at the same flow rate and adjusted to pH 4.0 with 1M HCl. Thesolution is then concentrated by ultrafiltration to 5% to 7% IgG and theionic strength is adjusted by diafiltration to 0.5 mS±0.2 mS. Finallythe solution is adjusted to 5.0%.

The solution is formulated as in Example 1. After sterile filtration,filling and capping, the solution is ready for delivery or storage. ACAfor different batches measured as defined above was found to be 0.5-0.7CH₅₀/mg immunoglobulin.

What is claimed is:
 1. In a method for producing IgG from plasma formedical applications, comprising: (i′) removing any albumin, resultingin an IgG fraction, (ii′) purifying IgG from said IgG fraction, byadsorbing IgG to a cation exchanger and collecting the adsorbed IgGfraction, and (iii′) inactivating any virus in said purified IgG whereinthe improvement comprises (I) concentrating the IgG fraction obtained instep (i′), (II) adjusting pH to 4±0.1 in the IgG released from thecation exchanger in step (ii′), and maintaining the pH below 6.0 duringthe remaining steps of the method; and (III) carrying out the virusinactivation (step iii′) by using chemicals at a temperature of 30°C.±2° C. for at least 4 hours.
 2. The method of claim 1 furthercomprising the steps: a) buffering of fresh plasma; b) removing anyeuglobulins; c) purifying of plasma fraction obtained after removal ofeuglobulins and albumin on an anion exchanger and collecting the unboundplasma fraction (IgG fraction); d) purifying of the IgG fractionobtained in step (c) on a cation exchanger and collecting the bound lgGplasma fraction including adjusting pH as in claim 1; e) concentratingthe IgG plasma fraction collected in step (d); f) inactivating any virusas defined in (III) of claim 1 in the IgG plasma fraction collected instep (e); g) removing any virus inactivation chemicals added in step (f)by adsorbing IgG to a cation exchanger and releasing and collecting thebound lgG plasma fraction; h) concentrating of the IgG plasma fractioncollected in step (g); i) formulating of the IgG plasma fractionconcentrated in step (h); and j) sterile filtering of the formulated IgGplasma fraction obtained in step (i).
 3. The method of claim 1, whereinthe concentration is performed by ultrafiltration to less than thevolume of the starting plasma.
 4. The method of claim 2, furthercomprising an adjustment of ionic strength to about 1.40 mS before stepg).
 5. The method of claim 2, wherein an acetate buffer is used in stepg).
 6. The method of claim 1, further comprising lowering after stepiii′ ionic strength to 0.5 mS±0.1.
 7. The method of claim 6, wherein thelowering of the ionic strength is by diafiltration against distilledwater.